Chemotherapy-induced peripheral neuropathy models constructed from human induced pluripotent stem cells and directly converted cells: a systematic review

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Introduction
The global incidence of cancer was estimated at 17 million in 2018 and is expected to further increase to 26 million by 2040. 64 large proportion of these patients will be treated with a regimen that consists of chemotherapy, among other treatment modalities.Yet, although the anticancer effects of chemotherapeutic treatment are undisputed, its side effects and sequelae can have a substantial impact on the quality of life of patients. 28hemotherapy-induced peripheral neuropathy (CIPN) is one of the most common long-term adverse effects of chemotherapy, occurring in up to 85% of patients, depending on the administered chemotherapeutic agent and dose. 68hemotherapy-induced peripheral neuropathy is characterized as a predominantly sensory neuropathy, manifesting as changes in sensation and (severe) neuropathic pain. 58,68Therefore, CIPN regularly leads to dose reduction or discontinuation of antineoplastic therapy.Although urgently needed, preventive strategies and treatment options for CIPN are currently limited. 13,58,68This is particularly troubling considering the expected increase in prevalence of CIPN, caused by improved cancer survival rates. 58aboratory studies investigating CIPN have historically been conducted using animal models, in part because of the inaccessibility of the human (peripheral) nervous system for research purposes. 11,13,38Although animal studies have provided valuable insights concerning CIPN, results have been difficult to translate to the human setting. 30,38This is particularly relevant to studies investigating potential analgesic treatment options because considerable genetic differences exist between humans and other species regarding the nociceptive system.For example, the expression levels and electrophysiological characteristics of prominent nociceptive markers differ substantially between small rodents and humans. 16,46onsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Meanwhile, progress in stem cell biology has led to the development of reprogramming and differentiation protocols that can be used to produce human nociceptive dorsal root ganglion (DRG) neurons from induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), and directly converted cells (DCCs). 29,53These techniques have since been adopted to model CIPN, resulting in the description of numerous laboratory models constructed from human neurons.Ultimately, these in vitro systems may be applied to investigate CIPN's pathophysiological mechanisms and explore pharmacological treatment strategies. 11However, published model descriptions vary widely in cellular setup and method of CIPN induction.This systematic review therefore provides a critical analysis of available models and their methodological considerations (ie, used cell type and source, CIPN induction strategy and validation method) for researchers willing to incorporate human in vitro models of CIPN in their research.

Search strategy
The protocol for this systematic review was prepared according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and prospectively registered with PROSPERO (CRD42021297136).The search strategy was developed with assistance from a clinical librarian.Search terms were related to iPSCs, ESCs, and DCCs and combined with keywords relevant for CIPN, using Boolean operators and truncation.Articles written in English and Dutch were considered for inclusion, whereas no restriction was placed on publication date.The literature search was performed on December 7, 2021, and updated on September 26, 2023, in MEDLINE (PubMed) and Embase (Ovid).The complete search is attached as Supplementary Material 1 (http://links.lww.com/PAIN/C11).

Eligibility criteria and study selection
Peer-reviewed experimental studies presenting original data and using relevant market available chemotherapeutic drugs were eligible for inclusion.
Considered articles had to describe development of CIPN models constructed from reprogrammed cells.Alternatively, studies that presented detailed information on outcome after treatment with chemotherapeutic agents were regarded as de facto model descriptions, and therefore also eligible for inclusion.Where applicable, intervention groups had to be compared with vehicle-treated or untreated neurons.Furthermore, studies had to mention outcome parameters relevant to model construction and validation, such as effects on neurite dynamics, cytotoxicity, and electrophysiological function. 11,27Studies that used only motor neuron cell culture systems were excluded.
Records were entered into Rayyan review software after removal of duplicates. 37Two researchers (P.S., K.H.) independently screened studies based on title and abstract and subsequently performed full-text screening.Thereafter, the reference lists of included studies were searched.Discrepancies were resolved between the 2 reviewers and, if needed, a third reviewer (N.W.).

Data extraction and presentation
Data concerning differentiated cell type and source, treatment (ie, agent, molar concentration, and treatment duration), and model validating assays was collected.Furthermore, to help guide investigators, we extrapolated the half-maximal inhibitory concentration (IC 50 ) of chemotherapeutic drugs, regarding cytotoxicity and neurite dynamics, from text, graphs, or tables, where possible.Methodological differences in incubation time and molar concentration were deemed too large for statistical pooling and appropriate conduction of meta-analysis.

Study selection
A total of 1095 unique records were found through database searches in MEDLINE (PubMed) and Embase (Ovid).Of these, 25 records were deemed appropriate for inclusion.Subsequent reference list searching of included articles identified 1 more eligible record.Thus, 26 articles were included in this systematic review.However, because 2 reports described the same experiments, these are hereafter consistently referenced as 1. 47,48 The flow chart of the literature search is depicted in Figure 1.Table 1 summarizes the included studies.

Aim of included studies
The included studies could be classified in 3 stages of complexity regarding their aim.The first group comprised of studies that performed elaborate neurotoxicity testing of different compounds but did not specifically list CIPN modelling as its aim (2 studies). 7,17The second group of studies aimed to model CIPN but stopped there (13 studies). 18,19,25,31,33,35,44,47,48,55,61,63,65,66,62 3.3.Methodological characteristics of studies

Reprogramming strategy and origin of neurons
The included publications reflect the advancements made in cellular reprogramming.As shown in Table 1, earlier studies mainly incorporated commercial cortical cell lines consisting of a mixed population of GABAergic and, to a lesser extent, glutamatergic neurons.Thereafter, models were constructed from pure populations of GABAergic neurons or commercial "peripheral" neurons.Two groups used both cortical and peripheral iPSC-derived cell lines and found higher sensitivity of peripheral cells to chemotherapy. 55,65Currently, most research groups incorporate peripheral nociceptive sensory neurons, primarily made using versions of the Chambers protocol, that aim to mimic nociceptive sensory DRG neurons. 3No publications were included that used co-culture of neurons and, for example, Schwann cells.
Three research articles describe the differentiation of either DCCs or ESCs, whereas all other articles used iPSCs as the starting material for experiments. 17,25,61,61

Antineoplastic treatment: drug, duration of treatment, and molar concentration
Chemotherapy-induced peripheral neuropathy models were constructed from a diverse group of antineoplastic agents (Table 2).Drug classes with the highest incidence of CIPN, that is, taxanes, vinca alkaloids, and platinum-based agents, were most represented.Bortezomib, a proteasome inhibitor, was also commonly studied.Numerous articles compared different drugs, but no combinations of drugs were tested in the same experimental setup.
Although there seems to be no consensus on the preferred treatment duration, all authors found that treatment of 24 to 72 hours was sufficient for induction of CIPN.,63,65,66 Justification for the chosen treatment duration was mostly absent and no obvious patterns could be observed for specific drug(s) (classes).Assays were performed immediately after completion of treatment for all but 2 publications.Snyder et al. evaluated treatment effects every 12 hours for the 72-hour treatment, whereas Schinke et al. were the only to test neurotoxicity (48 hours) after treatment had finished. 47,48,55Apart from the latter study, none report data regarding long-term effects of chemotherapeutic treatment within these model systems.
Drug concentrations were highly variable, even between studies using the same agent (Table 1).Most publications tested a range of molar concentrations, whereas some report applying concentrations based on in vivo plasma concentrations.Below we outline the effects of the 4 (paclitaxel, vincristine, bortezomib, and cisplatin) most used drugs.

Outcome parameters
A large number of different analyses were performed to assess the ability of chemotherapeutic agents to induce CIPN (Table 1).
Most applied were (immunofluorescence) imaging techniques assessing morphological changes to neurites (used as a surrogate marker of microtubule disruption) and assays studying cell viability or toxicity parameters.Three articles investigated intraneuronal transport (of mitochondria). 20,35,66Only 4 research groups assessed the electrophysiological properties of neuropathic neurons, using microelectrode array, whole-cell patchclamping, or calcium imaging. 8,18,19,65

Drug effects in model systems
Paclitaxel, vincristine, bortezomib, and cisplatin stood out for their frequent usage (Table 2).Therefore, we extrapolated the IC 50 of these drugs regarding neurite dynamics and cytotoxicity, in peripheral neurons.
Paclitaxel, a microtubule-stabilizing drug that prevents mitosis in cancer cells, was found to display a large difference in sensitivity for cytotoxicity and neurite dynamics (Fig. 2A).A similar profile was found for vincristine, another microtubule-targeting agent (Fig. 2B).Bortezomib, a drug that inhibits proteasomes resulting in ubiquitinated protein accumulation, showed an overlap between cytotoxicity and neurite impairment (Fig. 3A).This was also seen for platinum-based cisplatin (Fig. 3B).

Discussion
This systematic review includes 26 peer-reviewed studies using reprogrammed human cells to model CIPN.Reflecting the progress in human cellular reprogramming, we observed a shift in used cell type from cortical neurons to nociceptive DRG neurons.Models were almost exclusively validated by Different models of CIPN have been used over the last decennia.These include neuronal-like cell lines (eg, rat PC12 and neuroblastoma-derived SH-SY5Y), primary rodent DRG explants or dissociated cell cultures, and numerous animal (typically mice) in vivo models. 10,14,27Although these model systems have helped improve knowledge on the pathophysiological mechanisms of CIPN, no substantial gains have been made toward improvements in prevention and treatment of CIPN. 2,11This is often speculated to be due, in part, to the methodological and translational limitations of the current models.For example, the impaired reproducibility of primary cultures, genetic instability, and altered physiology of immortalized cell lines and unexpected variation in sensitivity to chemotherapy between different species and strains of animals all hinder clinical translation. 12,27,30,38,41Yet perhaps most importantly, inherent translational problems exist with results from animal studies because of genetic differences in the nociceptive system between humans and other species. 39,46,49,51,59Combined these issues indicate that (current) laboratory results interpreted as in vitro surrogate markers of clinical features of CIPN might not fully translate to human in vivo symptoms, such as neuropathic pain.Thus, an improved complementary human in vitro model of CIPN is needed.
Developments in human cellular reprogramming now allow for the creation of human nociceptive DRG neurons from unrelated cell types, such as umbilical cord blood cells. 3Thereby, the technology enables a method of disease modelling that overcomes many of the aforementioned problems. 24,29,52ecifically, strengths of human cellular reprogramming include its ability to produce theoretically unlimited quantities of otherwise inaccessible human cells, while concurrently providing more translatable results by enabling application of previously unattainable humanized in vitro models. 53Cellular reprogramming also offers the option of constructing patient-specific models using patients' cells, and important for CIPN modelling, acquired nociceptive DRG neurons correctly recapitulate chemotherapeutic substance specificity as observed clinically (eg, demonstrated by Schinke et al.). 24,47,48,53Yet the technique also has limitations.For example, terminal differentiation after reprogramming is  currently still a relatively expensive, time-consuming, and laborintensive process. 53Further, reprogramming could induce more variability than seen in primary DRG cultures, whereas the maturation status of obtained cells is another factor to consider. 50ncomplete differentiation could result in an impaired epigenetic status, whereas mature expression patterns of protein markers and correct electrophysiological functionality might require extended periods of culture. 42,45,67he studies included in this systematic review were the first to take advantage of this technique for CIPN.However, obvious from these reports is that modelling CIPN using reprogrammed cells is still in its infancy.Substantial heterogeneity between studies was visible in the way researchers applied chemotherapy and reported their findings.For example, paclitaxel concentrations ranged from 0.1 pM to 100 mM, although reasoning for the chosen concentration(s) was largely missing.Description of the reprogrammed cells also differed much between studies.Where some, such as Schinke et al., report the results of exhaustive morphological and functional characterization, others did not check the results of differentiation (Table 1). 47,4860,61 Furthermore, most of the reports validated treatment outcome solely by assessing the acute effects on neurite dynamics and cytotoxicity parameters, even though these may not be the only appropriate endpoint for antineoplastic drugs and should ideally be complemented by functional assays.This is exemplified by the fact that only 4 studies inspected the electrophysiological outcome of chemotherapeutic treatment. 8,18,19,65Additionally, sought after treatment effects were not prospectively formulated.Therefore, it is uncertain whether all the included studies were successful in recapitulating CIPN to its full extent.

Recommendations for future chemotherapy-induced peripheral neuropathy model development
Reliability and standardization of preclinical models, however, is crucial for their successful use in (pharmacological) research. 11,27ased on the results of this systematic review and expert opinion, we propose a number of suggestions to improve the clinical relevance and appropriateness of human cellular reprogramming-derived CIPN models.
First, we recommend the use of well-defined differentiated cell types and subsequent detailed description of differentiation protocols in publications.This should include reporting of a core set of cellular markers (eg, b3-tubulin, BRN3A, and TRPV1 in case of nociceptive DRG neurons).Cells may be of commercial origin; however, these are often not well characterized.Second, considering the impact of genetics on CIPN susceptibility and reprogramming-induced variability in iPSC cell lines, we suggest to use cells from multiple donors within the same project. 6,23,57hird, as the human DRG contains many different cell types, coculture of neurons with, for example, Schwann cells or macrophages might improve clinical translatability and should be considered. 23,26,34,36,40,69Also the application of threedimensional organoids may be of interest, however, their use in high-throughput (pharmacological) research is still limited by impracticalities, such as phenotypic variability and inaccessibility for microscopy assays. 23,69Fourth, future models should be validated using multiple methods that reflect the ongoing progress in pathophysiological knowledge.Importantly, this should include electrophysiological assays (eg, microelectrode array, whole-cell patch-clamping or calcium imaging) that can assess neuronal signal transduction as a surrogate maker of neuropathic pain. 1,56Fifth, because neuropathic effects may worsen after discontinuation of treatment with some antineoplastic agents, we advise to assess outcome also at later time points to factor in the phenomenon of coasting. 15,43Finally, to enhance transparency and facilitate the comparison of research results between laboratories, researchers should make their datasets accessible and include data availability statements in their manuscripts.

Strengths and limitations
This systematic review adhered to a prospectively registered protocol to ensure transparency and increase reproducibility.Furthermore, we followed the PRISMA guidelines and performed independent record screening and data extraction.A clinical librarian was consulted to help develop the search strategy and maximize relevant search results.Combined these methodological strengths explain why this study was able to extract twice as many high-quality reports than another recently published systematic review. 32ble 2 Overview of used chemotherapeutic agents.September 2024 • Volume 165 • Number 9 www.painjournalonline.com The main limitation of this study relates to the heterogeneity of the individual reports, in both model construction and reporting.Conduction of meta-analysis was deemed inappropriate and, thus, only minimal guidance can be offered regarding drug concentrations.To combat this, we extracted and plotted the IC 50 s of the most frequently used drugs; however, for some studies we were only able to provide estimates.Differing quality of reporting also made it difficult to properly assess the internal validity of studies; yet, they seem to be largely in agreement with each other regarding cytotoxicity and neurite outgrowth endpoints (Figs. 2 and 3).

Conclusions
This systematic review describes the first studies that used human cellular reprogramming techniques to model CIPN.Considerable differences in methodology were found, thus limiting comparability and translatability of studies.Therefore, a number of recommendations are provided for prospective researchers interested in modeling CIPN.Ultimately, these humanized models hold the potential to enlarge pathophysiological knowledge and aid in the discovery of novel intervention strategies.

Figure 1 .
Figure 1.Flowchart of the literature search.

Figure 2 .
Figure 2. Peripheral neurons show differential sensitivity regarding cytotoxicity and neurite dynamics for paclitaxel and vincristine.Data points show the extrapolated half-maximal inhibitory concentrations (IC 50 ) for paclitaxel (A) and vincristine (B).The results of the most mature, best-defined cell line and shortest treatment duration are depicted for studies that tested multiple strategies.Only assay-based results are shown for cytotoxicity.Importantly, IC 50 s of chemotherapeutic agents might be dependent on factors (ie, specific assay and cell line, treatment duration, and cell age) that could not, or only partially, be accounted for in this figure.Data were extrapolated from text, graphs, and tables.

Figure 3 .
Figure 3. Bortezomib and cisplatin have an overlapping effect on cytotoxicity and neurite dynamics in peripheral neurons.Data points show the extrapolated halfmaximal inhibitory concentrations (IC 50 ) for bortezomib (A) and cisplatin (B).The results of the most mature, best-defined cell line and shortest treatment duration are depicted for studies that tested multiple strategies.Only assay-based results are shown for cytotoxicity.Importantly, IC 50 s of chemotherapeutic agents might be dependent on factors (ie, specific assay and cell line, treatment duration, and cell age) that could not, or only partially, be accounted for in this figure.Data were extrapolated from text, graphs, and tables.

Table 1
Overview of included studies.